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Antimicrobial Agents and Chemotherapy, August 2008, p. 2742-2749, Vol. 52, No. 8
0066-4804/08/$08.00+0     doi:10.1128/AAC.00235-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Differential Expression of the Smb Bacteriocin in Streptococcus mutans Isolates {triangledown}

Hideo Yonezawa,1 Howard K. Kuramitsu,2 Shu-ichi Nakayama,1 Jiro Mitobe,1 Mizuho Motegi,1 Ryoma Nakao,1 Haruo Watanabe,1 and Hidenobu Senpuku1*

Department of Bacteriology, National Institute of Infectious Diseases, Tokyo, Japan,1 Department of Oral Biology, State University of New York at Buffalo, Buffalo, New York2

Received 20 February 2008/ Returned for modification 24 March 2008/ Accepted 12 May 2008


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ABSTRACT
 
The two-component lantibiotic Smb is produced by Streptococcus mutans GS5. In the present study, we identified seven strains of S. mutans containing the smb gene cluster. These strains could be classified into high- and low-level Smb producers relative to the levels of Smb production by indicator strains in vitro. This classification was dependent upon the transcription levels of the structural smbA and smbB genes. Sequence analysis upstream of smbA in the high- and low-level Smb-producing strains revealed differences at nucleotide position –46 relative to the smbA start codon. Interestingly, the transcription start site was present upstream of the point mutation, indicating that both groups of strains have the same promoter constructs and that the differential expression of smbA and smbB mRNA occurred subsequent to transcription initiation. In addition, smbA::lacZ fusion expression was higher when it was regulated by the sequences of strains with high-level Smb activity than when it was regulated by the comparable region from strains with low-level Smb activity. Taken together, we conclude that high- or low-level Smb expression is dependent on the presence of a G or a T nucleotide at position –46 relative to the smbA translational start site in S. mutans Smb producers.


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INTRODUCTION
 
Streptococcus mutans, the principal etiological agent of human dental caries, is present along with other oral bacteria in heterogeneous biofilms termed dental plaque (7). Since different strains of S. mutans have been shown to produce bacteriocins, also termed mutacins, some of which are active against other oral bacteria (16). One of them, Smb, which is regulated by a competence-stimulating peptide (CSP)-dependent quorum-sensing system, is a two-component lantibiotic produced by S. mutans strain GS5 (16, 24). The biosynthetic apparatus of lantibiotics is generally organized in gene clusters (20); and the smb operon consists of seven open reading frames (ORFs) in the order smbM1, -F, -T, -M2, -G, -A, and -B, flanked by putative transposase genes (24). The smbF and smbG genes are thought to be involved in immunity to Smb. A recent report also indicated that the smbG gene appears to play a role in the sensitivity of strain GS5 to a variety of antimicrobial agents, such as tetracycline and triclosan (10). The smbT gene is presumably the ATP binding cassette transporter for pre-Smb processing and secretion. The structural genes for the precursor preproSmb consist of smbA and smbB. These genes encode the two peptides SmbA and SmbB, respectively, within a single operon (16, 24). The two-component lantibiotic systems utilize two peptides that are each posttranslationally modified to an active form and that act in synergy to produce antibacterial activity (3). The gene cluster encoding Smb expression also encodes two putative modification enzymes, which have been designated smbM1 and smbM2 (24). Recently, predicted lanthionine and methyllanthionine ring forms were proposed for other known two-component lantibiotics similar to Smb (11).

S. mutans is thought to use Smb production as a means to compete with other oral bacteria present in dental plaque (22). Some reports indicated that the bacteriocins produced by this organism play an important role in the regulation and composition of dental biofilms (7, 9, 14). On the other hand, some reports failed to show a relationship between the presence of bacteriocin genes in different strains of this organism and caries status (4, 6). In the present study, we demonstrate that a range of Smb activities are present in S. mutans clinical isolates harboring the smb operon. In addition, we describe evidence that a single base mutation in the upstream region of smbA can account for these differences.


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MATERIALS AND METHODS
 
Bacterial strains. The S. mutans strains used in this study are listed in Table 1. S. mutans wild-type strains were assessed for the presence of the smb genes as well as the expression of Smb activity. RP66 (group C streptococcus, which is sensitive to Smb) and oral streptococci (Streptococcus sanguinis ATCC 10556, ST205, and ST134; Streptococcus mitis ATCC 903 and ATCC 6249; Streptococcus gordonii ATCC 10558 and Challis; and Streptococcus salivarius HT9R, JSM5707, and ATCC 9759) were used as indicator strains for Smb activity. These strains were grown in brain heart infusion (BHI) medium (Difco Laboratories, Detroit, MI) in an anaerobic atmosphere of 85% N2, 10% CO2, and 5% H2 at 37°C. Transformants of S. mutans were selected following their growth on BHI agar plates supplemented with 10 µg of erythromycin per ml or 500 µg of kanamycin per ml.


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  		TABLE 1. Bacterial strains and plasmids

Agar plate bacteriocin assays. Loopfuls of stationary-phase cultures of S. mutans strains were stabbed into BHI agar plates. The plates were incubated at 37°C for 16 h. Indicator strains were grown to an optical density of 0.2 at 550 nm. Each culture was then diluted 1:100, and 0.2 ml of this solution was pipetted into a tube containing 4 ml of molten BHI broth containing 1% agar. This solution was mixed and poured evenly onto the surfaces of the plates, the plates were incubated at 37°C for an additional 24 h, and the diameters of the zones of inhibition were measured.

Construction of smbAB and nlmAB mutants. The mutants with defective smbAB genes were constructed by double-crossover homologous recombination by insertion of an erythromycin resistance determinant into the genes, as described previously (24). In addition, the nonlantibiotic mutacin (19) nlmAB mutant was also constructed by the same method. Confirmation that plasmid insertion caused gene disruption was determined either by Southern blotting or by PCR.

Extraction of RNA, real-time quantitative RT-PCR, and primer extension analysis. The S. mutans strains were grown on BHI agar plates. After incubation at 37°C 10 h, the cells were scraped from the plates and resuspended in phosphate-buffered saline (PBS). After centrifugation and washing of the cells with PBS three times, the cells were suspended in 0.3 ml of diethylpyrocarbonate-treated water. RNA extraction was carried out as described previously (24). The RNA samples were then treated for 15 min at 37°C with 1.0 U of RNase-free DNase (Amersham Biosciences Corp., Piscataway, NJ) per ml to remove contaminating DNA. Reverse transcription (RT) was carried out with a SuperScript III kit (Invitrogen, Corp., Carlsbad, CA), according to the directions of the supplier. The real-time RT-PCR was performed with cDNA samples with either the 16S rRNA-specific primers (primers LARNA5 and LARNA6 as internal controls) or smb-specific primers (SmbABRTFw and SmbABRTRev) with Power SYBR green PCR master mixture (Applied Biosystems, Foster City, CA) in an ABI Prism 7700 sequence detection system (Applied Biosystems). The final results were expressed as the level of smb gene expression relative to the level of 16S rRNA gene expression, i.e., relative expression = level of smbAB gene expression/level of 16S rRNA gene expression. A similar approach was used for the smbG to smbA transcript (with primer pair SmbGARTFw and SmbGARTRev).

Total RNA was prepared as described above, and primer extension was carried out essentially as described previously (13). RNA (20 µg) was annealed to 2.0 pmol of [{gamma}-32P]ATP-labeled primer SmbAPE (Table 2; see also Fig. 3A) at 80°C for 2 min and then at 37°C for 45 min in RT buffer (Omniscript RT kit; Qiagen, Valencia, CA). Then, 0.5 mM of the four deoxynucleoside triphosphates and reverse transcriptase from an Omniscript RT kit were added to 60 µl of the solution. The primer extension reaction was done at 37°C for 1 h. The synthesized DNA was extracted with phenol-chloroform, precipitated with ethanol, dissolved in DNA-sequencing load buffer (SequiTherm Excel II DNA sequencing kit; Epicentre Biotechnologies, Madison, WI), and analyzed by electrophoresis on an 8% polyacrylamide gel containing 50% urea, followed by autoradiography. As a standard, the same 32P-labeled primer was annealed to alkaline-denatured DNA from pSmbABH, and the dideoxy chain termination sequencing reaction was carried out with the SequiTherm Excel II DNA sequencing kit. The sample was loaded on the gel along with the synthesized DNA described above.


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  		TABLE 2. Oligonucleotide sequences of PCR primers


Figure 3
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  		FIG. 3. (A) Sequence alignment of the upstream regions of smb-containing S. mutans strains. The uppermost sequence is that corresponding to the smbG C-terminal sequence, and the bottom sequence is that of the regions flanking the smbB gene derived from strain GS5 Smb (GenBank accession no. AB179778). The boxes indicate identical sequences in all strains. The primers for the PCR amplification of this region are marked with arrows under their nucleotide sequences. Dashed lines under these sequences denote the smbA or smbB ATG start codon. Double dashed lines denote the smbG, smbA, or smbB stop codon. The numbers –1, –46, and –60 indicate the position relative to the start codon of smbA. At the top, the circle with the arrow indicates the mapped transcription initiation site of smbA. The heavy bars represent the putative –10 and –35 regions of a candidate promoter region. (B) Primer extension analyses confirmed the 5' end of the smbA transcript.

Construction of upsmbA::lacZ fusion plasmids. To construct a fusion fragment of the upsmbA region and lacZ, the upstream regions of smbA containing the promoter regions from strain FSM-6 or BM71 and the lacZ gene from the pSV-beta-galactosidase control vector (Promega, Madison, WI) were used as templates with each pair of specific primers, each of which contained a 9- or 14-bp tag fragment (Table 2). The first step of PCR amplification was performed with each template and each primer pair (primers UpsmbAFwBam and UpsmbARevTag or primers LacFwTag and LacRevXba). After amplification, the 183-bp upsmbA region and the 3,285-bp lacZ amplification fragments were treated with a PCR cleanup kit (Qiagen GmbH, Hilden, Germany) and were used as templates. In addition, the amplified fragments served as primers for the amplification of each other. The secondary PCR mixture contained 1 µl of each PCR fragment, 5 µl of 10x PCR buffer (Takara Shuzo, Tokyo, Japan), 200 mM MgCl2, and 1.25 U of high-fidelity DNA polymerase Pyrobest (Takara Shuzo). The temperature profile included, first, denaturation at 94°C for 5 min, followed by 15 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 3 min and then heating at 72°C for 5 min. The 3,454-bp amplified fragments were purified and used as templates for the third PCR with primers UpsmbAFwBam and LacZRevXba. The amplified 3,454-bp PCR fragments were digested with BamHI and XbaI and ligated into the BamHI-XbaI sites of Escherichia coli-Streptococcus shuttle vector pDL276 (2). The plasmids containing the upsmbA-lacZ fusions were then transformed into E. coli DH5{alpha} and the transformants were selected on LB agar plates containing 50 µg of kanamycin per ml. The relevant structures of the resultant plasmids, named pSmblacL and pSmblacH, were confirmed by DNA sequencing.

Determination of β-galactosidase activity. Plasmids pSmblacL and pSmblacH and control plasmid pDL276 were transformed into S. mutans strain FSM-6 or BM71. Transformants of the S. mutans strains were selected on mitis salivarius agar plates containing 500 µg of kanamycin per ml. For measurement of β-galactosidase activity, each transformant was incubated on a BHI agar plate containing kanamycin. After incubation at 37°C for 10 h, the cells were scraped from the agar plate and resuspended in PBS. The cells were centrifuged and assayed for β-galactosidase activity, as described previously (5).

In vitro mutagenesis. A PCR-based oligonucleotide-directed mutagenesis strategy was carried out for site-directed mutagenesis of the smbA promoter region. The synthetic oligonucleotides used as mutagenic primers are listed in Table 2. Briefly, plasmid pSmblacL was used as a template for mutagenesis with primer pair SiteTtoG and SiteTtoGcomp. Plasmid pSmblacH was also used as a template in mutagenesis with primer pair SiteGtoT and SiteGtoTcomp. The PCR products were treated with the restriction enzyme DpnI (New England Biolabs, Beverly, MA), and the treated products were transformed into E. coli DH5{alpha}. The mutagenic plasmids were then subjected to DNA sequencing to confirm that the desired mutation but no additional mutation was present (data not shown). These mutagenized plasmids, named pSmblacLAGC and pSmblacHATC, respectively, were transformed into S. mutans strain FSM-6 or BM71, and the transformants were used for β-galactosidase activity assays.

Construction of smbA and smbB expression vectors. To construct the vectors expressing the smbA and smbB genes, the smbA and smbB regions downstream from the upsmbA region were amplified with primer pair UpSmbAFwBam and RevSmbABKpn (Table 2). The chromosomal DNA from strain FSM-6 or BM71 was used as the template. The amplified 573-bp fragments containing the BamHI and KpnI sites were then digested with BamHI and KpnI and ligated into the BamHI and KpnI sites of pDL276 (2). The plasmids containing the upsmbA-smbB region were then transformed into E. coli DH5{alpha} and the transformants were selected on LB agar plates containing 50 µg of kanamycin per ml. The relevant structures of the resultant plasmids, named pSmbABL (with fragments amplified from strain FSM-6) and pSmbABH (with fragments amplified from BM71), were confirmed by DNA sequencing. These plasmids were transformed into the S. mutans BM71 smbAB mutant, and the transformants were used for bacteriocin agar plate assays with the S. salivarius strain.


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RESULTS AND DISCUSSION
 
Identification of smb genes in S. mutans clinical isolates. By screening 17 clinical isolates of S. mutans (12) by PCR and Southern blotting for the presence of the smb genes, we found five positive strains (strains FSC-4, FSM-6, FSC-8, FSC-1, and FSM-3). In addition, laboratory strains GS5 and BM71, which are known to be producers of Smb, were confirmed to harbor these genes (22, 24). We previously determined that the smb operon consists of seven ORFs (24). We observed that all of these positive strains possess all of the ORFs by PCR or Southern blotting analysis (data not shown). In order to assess the antimicrobial activities of these Smb-producing strains, bacteriocin agar plate assays were performed with some potential indicators strains (the strains are noted in the Materials and Methods section). S. mutans strain BM71 exhibited the maximum inhibitory zones against most of the indicator strains (data not shown). Variations in the antimicrobial activities of the S. mutans strains against these indicator strains were also observed. The most uniform results, separation into high- and low-level Smb-producing groups, was accomplished with S. salivarius JCM5907 as an indicator strain in the bacteriocin agar plate assays (Fig. 1A). Strains GS5, BM71, FSC-1, FSM-3, and FSM-6 exhibited bacteriocin activity; on the other hand, strains FSC-4 and FSC-8 did not produce inhibitory zones.


Figure 1
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  		FIG. 1. (A) S. mutans production of agents with activity against S. salivarius strain JCM5907 as an indicator strain determined by bacteriocin agar plate assays; (B) activities of S. mutans wild-type (WT) strain FSM-6 (lane 1) and its smbAB mutant (lane 2) against S. salivarius determined by bacteriocin agar plate assays; (C) activities of wild-type strain BM71 (lane 1), its nlmAB mutant (lane 2), as well as its smbAB mutant (lane 3) against S. salivarius strain JCM5907.

Previous reports indicated that some of the S. mutans strains possess two or more bacteriocins (7, 19, 21). To determine whether these Smb producers express other bacteriocins (mutacins I to IV) (1, 17, 18, 19), PCR analysis, Southern blot analysis, and sequence examination were carried out. We determined that the mutacin I genes (18) were present in the genome of strain FSM-6 (data not shown). Mutacin I belongs to the lantibiotic family and has a high level of activity against oral streptococci (7, 18, 19). We disrupted the smbAB genes in FSM-6 by inserting an erythromycin cassette within the genes and examined its antimicrobial activity with S. salivarius strain JCM5907 (Fig. 1B). The smbAB mutant of strain FSM-6 was as inhibitory as the parental strain against the indicator strains. We determined the transcription levels of the smbAB genes and found that the level of expression was low (Fig. 2), as described in more detail below. These results suggest that the bacteriocin activity of FSM-6 against the S. salivarius strain might be due to the expression of mutacin I or another antimicrobial agent.


Figure 2
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  		FIG. 2. Expression of smbA to smbB (A) and smbG to smbA (B) transcription by S. mutans strains. The quantity of cDNA corresponding to these genes was determined by real-time RT-PCR and was normalized to that of the 16S rRNA gene in each unique reaction. Each experiment was repeated three times with duplicate samples from each independently isolated RNA preparation. Data are expressed as the means of all of experiments ± standard deviations.

We further determined that strains FSC-1, GS5, and BM71 possessed the nonlantibiotic mutacin IV (data not shown), which has been classified as a nonlantibiotic bacteriocin encoded by the nlmA and the nlmB genes (24). To determine which bacteriocin is required for inhibition of the growth of the S. salivarius indicator strain, the levels of bacteriocin production by smbAB and nlmAB mutants of these strains were compared. We attempted several times to construct an smbAB mutant of FSC-1. However, we were not successful, since this strain apparently is not genetically competent. Therefore, the zones of inhibition of strains BM71 and GS5 and their mutants in plate assays with the indicator strain were compared. Both smbAB mutant strains were completely devoid of bacteriocin activity against the indicator strain, while the mutation in the nlmAB genes had no influence on inhibition of the growth of the S. salivarius strain compared to that of their parental strains (Fig. 1C, strain BM71). These results indicated that Smb, but not mutacin IV, is the major bacteriocin in these strains that is active against the S. salivarius strain. A recent study has also indicated that Smb, but not mutacin IV, was isolated from the GS5 culture medium (16). This suggests that strain GS5 might not express mutacin IV under normal laboratory conditions.

Other mutacin genes are not present in the genomes of several Smb producers (strains FSM-3, FSC-4, and FSC-8). Strains FSC-1 and FSM-3 exhibited moderate inhibitory zones relative to those for strains GS-5 and BM71 (Fig. 1A). The growth of strains FSC-1 and FSM-3 was relatively slow, and their final titers in liquid culture, as determined from the optical density at 600 nm, were approximately 55% of the titers of strains GS5 and BM71. These results suggest that the relatively slower growth and final cell numbers of strains FSC-1 and FSM-3 may be responsible for the apparent relatively modest Smb activity of these strains. Although we did not obtain direct evidence for the role of Smb in inhibiting the growth of the S. salivarius indicator strain by strain FSC-1, we concluded that strain FSC-1 expresses Smb activity at levels similar to those for strains GS5 and BM71 under these conditions. Therefore, these results suggested that the Smb producer strains could be divided into high-level producers (strains GS5, BM71, FSC-1, and FSM-3) and low-level producers (strains FSC-4, FSM-6, and FSC-8) of Smb activity.

RT-PCR analysis of smbAB transcription. We hypothesized that there were sequence differences in the smb structural genes between the groups with high- and low-level Smb activity. Therefore, we analyzed the sequences of the smbA and smbB genes from the two groups. However, there were no differences in the smbA and smbB gene sequences of strains FSC-4, FSM-6, and FSC-8 compared with the comparable sequences in high-level Smb-producing strain GS5. We then determined if there were differences in the levels of transcription of the smbA and smbB genes between the two groups using RT-PCR. A comparison of smbA and smbB gene expression in these strains (Fig. 2A) revealed that the expression of both genes was significantly elevated in the strains with higher levels of activity than in the strains with lower levels of activity.

We previously reported that there is a single smb operon with two promoters in strain GS5. One is upstream of smbM1, which is the first gene of the smb gene cluster, and the other is immediately upstream of smbA and has a terminator sequence downstream of smbB (24). This suggests that smbA and smbB transcription might be regulated by one or two promoters in the smb operon. In order to analyze the effects of the promoter upstream of the smbM1 gene on the expression of smbA and smbB, real-time RT-PCR was carried out with primer pair SmbGARTFw and SmbGARTRev (Table 2 and Fig. 3A), which detects the transcript from smbG to smbA (Fig. 2B). No difference in the level of expression of this transcript was detected between strains in the two Smb expression groups. Since this transcript is regulated by the promoter immediately upstream of the smbM1 gene (24), this promoter does not appear to be responsible for the differential expression of Smb in the two groups. However, the levels of transcription of the smbA and smbB genes in all strains were higher than those of smbG and smbA (Fig. 2). Furthermore, there was a clear difference in the levels of transcription of the smbAB genes between the groups with high and low levels of Smb activity. These results suggest that the promoter region immediately upstream from the smbA gene is primarily responsible for the differential expression of these two genes in the two groups.

Sequence analysis of smbA promoter region. In order to determine whether sequence differences in the smbA promoter region are responsible for the differential expression of the Smb structural genes in the two groups, sequence analysis of this region was carried out (Fig. 3A). Sequencing of this region of the strains with high and low levels of Smb activity revealed the presence of a transversion mutation (G to T) at nucleotide position –46 relative to the smbA ATG start codon in the strains with low levels of Smb activity (strains FSC-4, FSM-6, and FSC-8) compared with the sequences of the strains with high levels of Smb activity, including laboratory strain BM71, which is a reference strain for Smb in the GenBank nucleotide sequence database (GenBank accession no. AB179778), as well as strain GS5. No other sequence differences were detected in this region, which is partially shown in Fig. 3A, in any of the strains. These results indicate that a G nucleotide at position –46 results in a relatively higher level of expression of the smbAB genes compared with the level of expression when a T nucleotide is present at this position. We next analyzed the smbA and smbB transcripts by primer extension mapping in order to determine the transcription start site. The template RNAs were extracted from the groups with high (strain BM71) and low (strain FSM-6) levels of Smb antimicrobial activity and were used for the experiment with primer PEUpsmbA (Table 2 and Fig. 3A). The results of the mapping are shown in Fig. 3B. We observed a single major extended band at the same position in both strains. Comparison of the band with a DNA sequencing ladder as a standard showed that the 5' end of the mRNA corresponded to the T nucleotide at position –81 bp relative to the start codon of smbA, which is within the smbG ORF (Fig. 3A). The putative candidate of the promoter structure is shown in Fig. 3A (–35 sequences are ATGTAT, and –10 sequences are TATTGA, as indicated by the heavy bars). The intensities of the bands from the primer extension reaction with RNA from strain BM71 were much higher than those with RNA from FSM-6, although the same amount of RNA from each strain was used for this reaction (compare lanes 1 and 2 in Fig. 3B). This is consistent with the observation of the RT-PCR results (Fig. 2A). In this promoter region, all strains contained the same sequences, indicating that all of the strains have the same promoter constructs and that the events which discriminate between the strains with high and low levels of Smb activity likely occurred after the transcription starts. To explain these differences, we first considered that the regulation of mRNA transcription might be dependent upon a decrease in the stability of the mRNA of the smbA and smbB genes in the group with a low level of Smb activity as a result of the nucleotide transversion. However, there were no detectable differences in mRNA stability between the high- and low-level Smb producers following analysis of the cDNA synthesized from rifampin-treated cells (data not shown). Next, we hypothesized that the transversion may involve a palindrome construct. Indeed, the prediction obtained by sequence analysis with Genetyx (version 7.0.3) software suggested that a G-to-T nucleotide change might result in a higher probability of formation of a palindromic sequence in this region, suggesting that this construct might decrease the possibility that the gene will proceed with subsequent transcription. There is also a possibility that the transversion region may be an operator region which serves as a binding site for a regulatory protein(s) that has yet to be identified. Further research is now in progress to examine the mechanism of this regulation.

Characterization of the smbA flanking region and its influence on smbA and smbB translation. In order to confirm the effects of nucleotide changes at position –46 on the translation of the smbA and smbB genes, the primer structure of low-level Smb-producing strain FSM-6 and high-level Smb-producing strain BM71 were fused to the promoterless lacZ gene of pDL276 to produce chimeric plasmids pSmblacL and pSmblacH, respectively. These plasmids were transformed into strains FSM-6 and BM71, and the strains were assayed for their β-galactosidase activities (Fig. 4A). For strains harboring pSmblacL (strains FSM-6-lacL and BM71-lacL) or pSmblacH (strains FSM-6-lacH and BM71-lacH), the results clearly indicated that the β-galactosidase activities of strains FSM-6-lacH and BM71-lacH were significantly higher than those of strains FSM-6-lacL and BM71-lacL, respectively (β-galactosidase activity of strain BM71-lacH, 850 ± 72 U).


Figure 4
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  		FIG. 4. (A) β-Galactosidase activities of strains BM71-lacL and BM71-lacH, strains FSM-6-lacL and FSM-6-lacH, and strains BM71-lacHATC and BM71-lacLAGC. The activity (percent) of each strain was calculated by use of the activity of strain BM71-lacH, which was set equal to 100%. Strains BM71-lacHATC and BM71-lacLAGC were generated from the BM71 smbAB mutant with plasmids pSmblacH and pSmblacL, respectively, following site-directed mutagenesis, as described in the text. The experiments were repeated three times with duplicate samples. Data are expressed as the means of all of experiments ± standard deviations. (B) Bacteriocin activity of the BM71 smbAB mutant complemented with plasmids carrying the smbAB region from strains with low and high levels of Smb activity. The bacteriocin activity was measured by the agar plate assay with S. salivarius strain JCM5907. smbA- and smbB-containing upsmbA was amplified from BM71 (lane 1) or FSM-6 (lane 2), and the constructs were transformed into the BM71 smbAB mutant.

To confirm the effect of the nucleotide transversion on the level of Smb expression, site-directed mutagenesis of these two plasmids was carried out. In plasmid pSmblacL, the T nucleotide at position –46 was changed to G. Likewise, the G nucleotide in pSmblacH was changed to a T in the comparable flanking region (named pSmblacLAGC and pSmblacHATC, respectively), and strain BM71 with these plasmids (BM71-lacLAGC and BM71-lacHATC, respectively) was tested for β-galactosidase activity. The β-galactosidase activities of BM71-lacL and BM71-lacHATC were similar. Furthermore, the activities of BM71-lacH and BM71-lacLAGC also covered a similar range (Fig. 4A), indicating that the difference in the levels of gene expression at both the transcriptional and the translational levels is dependent upon the single base mutation at nucleotide position –46. In addition, these results indicate that nonspecific factors (i.e., unexpected mutations) did not occur in the plasmids.

Expression of smbA and smbB genes in the BM71 smbAB mutant. In order to directly verify that the Smb-positive phenotype is influenced by the G nucleotide at position –46, the smbA and smbB gene expression plasmid, named pSmbABH, was constructed by using the fragment amplified from strain BM71. In addition, Smb-negative phenotype expression vector pSmbABL was also constructed with FSM-6. These plasmids were introduced into the BM71 smbAB mutant, and the Smb activities of these strains were analyzed by the bacteriocin agar plate assays with the S. salivarius indicator strain (Fig. 4B). The BM71 smbAB mutant was completely devoid of bacteriocin activity (Fig. 1C). In the equivalent strain containing plasmid pSmbABH, the Smb activity was restored to the wild-type level. In contrast, the complemented strain harboring pSmbABL did not have enhanced bacteriocin activity. These results indicate that the nucleotide at position –46 upstream of the smbA gene directly influences Smb activity via the transcription of the smbA and smbB genes.

In conclusion, we characterized the differential antimicrobial activities of Smb in S. mutans strains. The Smb-positive strains were shown to possess all of the smb ORFs. The differential expression of the smbA and smbB genes resulted from a single nucleotide transversion. Although a recent report suggested a relationship between mutacin production and caries status (4, 6), statistical analysis failed to show a positive correlation. However, this analysis was based upon the genotypes of the bacteriocins. Our present results suggest that it is important to assess not only the genotypes of the bacteriocins but also the actual antimicrobial activities of the bacteriocins. The production of high levels of bacteriocin, including Smb, may kill some neighboring streptococcal species, and subsequently, S. mutans may predominate within dental plaque, leading to dental caries. Moreover, Smb production may induce the release of DNA from killed competing streptococci. The liberated DNA could then function as a component of a biofilm structure (8, 15). Subsequently, increased biofilm formation could also induce the expression of additional Smb via S. mutans quorum-sensing regulation (16, 23, 24, 25). However, the relationship between Smb production by S. mutans and caries status remains to be determined.


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ACKNOWLEDGMENTS
 
We acknowledge Makoto Ohnishi, Ken Shimuta, Tomoko Hanawa, Tadayoshi Ikebe, Yosuke Tashiro, and Nobuhiko Nomura for their technical support and helpful discussion.

This work was supported in part by grants-in-aid for Development Scientific Research (grants 15390571 and 19659559) from the Ministry of Education, Science, and Culture of Japan and the Ministry of Health, Labor, and Welfare (grants H16-Medical Services-014 and H19-Medical Services-007).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bacteriology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111. Fax: 81-3-5285-1163. E-mail: hsenpuku{at}nih.go.jp Back

{triangledown} Published ahead of print on 19 May 2008. Back


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Antimicrobial Agents and Chemotherapy, August 2008, p. 2742-2749, Vol. 52, No. 8
0066-4804/08/$08.00+0     doi:10.1128/AAC.00235-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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